Inducible Nitric Oxide Synthase Binds, S-Nitrosylates, and Activates Cyclooxygenase-2

ABSTRACT

Cyclooxygenase (COX2) and inducible nitric oxide synthase (iNOS) are two major inflammatory mediators. Inducible NOS specifically binds to COX2 and S-nitrosylates it, enhancing COX2 catalytic activity. Selectively disrupting iNOS-COX2 binding prevents NO-mediated activation of COX2. The synergistic molecular interaction between two inflammatory systems permits assays for developing anti-inflammatory drugs.

This application claims the benefit of provisional application Ser. No.60/675,552 filed Apr. 28, 2005, the disclosure of which is expresslyincorporated herein.

The invention was made using funds from the U.S. government, grantnumbers DA00266 and DA00074 from NIDA. Therefore, the U.S. governmentretains certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of drug screening andtherapeutics. In particular, it relates to anti-inflammatory agents.

BACKGROUND OF THE INVENTION

Cyclooxygenase-2 (COX2; prostaglandin H₂ synthase) and inducible nitricoxide (NO) synthase (iNOS) are two of the principal inflammatorymediators. (1, 2). Following inflammatory stimuli, these two enzymes,which are inactive under basal conditions, undergo new synthesis,respectively manufacturing large quantities of prostaglandins and NO.Needleman and associates had shown that NOS inhibitors can prevent theformation of prostaglandins (Salvemini et al., PNAS 90:7240, 1993). Thesimplest interpretation of these findings would be that NO, a freeradical, gives rise to even more toxic free radicals such asperoxynitrite and hydroxide free radical, which constitute inflammatorystimuli that would lead to induction of COX2 and formation ofprostaglandins.

COX2 inhibitors have attained widespread use as anti-inflammatoryagents, though they elicit potentially adverse side effects (1, 3-5),while iNOS inhibitors are not presently employed therapeutically.Inflammatory stimuli elicit new synthesis of iNOS and COX2 proteins withsimilar time courses suggesting that the two systems may interact(6, 7).Stimulants of iNOS such as bradykinin (8) and lipopolysaccharide (LPS)plus interferon y (IFNγ), components of endotoxin, also enhanceprostaglandin formation (9).

There is a continuing need in the art to develop therapies for reducingdisease conditions caused by or exacerbated by inflammation.

SUMMARY OF THE INVENTION

According to one embodiment of the invention a method is provided forscreening for substances useful for relieving inflammation. A testsubstance is contacted with a first protein and a second protein underconditions in which the first protein and the second protein bind toeach other. The first protein is selected from the group consisting ofmammalian iNOS, a fragment of mammalian iNOS from the N-terminal 10% ofiNOS sufficient to bind COX2, and a fusion protein comprising saidfragment of mammalian iNOS. The second protein is selected from thegroup consisting of mammalian COX2, a fragment of mammalian COX2 fromthe C-terminal 20% of COX2 sufficient to bind iNOS, and a fusion proteincomprising said fragment of COX2. The amount of free or bound of saidfirst or said second protein is determined. A test substance isidentified as a candidate drug for relieving inflammation if itincreases the amount of free first or second protein or decreases theamount of bound first or second protein.

Another aspect of the invention is a method of treating inflammation ina patient. An antibody which binds to amino acid residues 1-114 ofmammalian iNOS or amino acid residues 484-604 or 488-604 of mammalianCOX2 is administered to the patient. Binding of iNOS to COX2 is therebyinhibited in the patient.

Yet another embodiment of the invention is a method of treatinginflammation in a patient. A nucleic acid vector encoding a polypeptidecomprising a fragment of human iNOS from the N-terminal 10% of iNOSsufficient to bind COX2 or a fragment of human COX2 from the C-terminal20% of COX2 sufficient to bind iNOS, is administered to the patient.Binding of iNOS to COX2 is thereby inhibited in the patient.

A further aspect of the invention is a method of treating inflammationin a patient. A polypeptide comprising a fragment of human iNOS from theN-terminal 10% of iNOS sufficient to bind COX2 or a fragment of humanCOX2 from the C-terminal 20% of COX2 sufficient to bind iNOS, isadministered to the patient. Binding of iNOS to COX2 is therebyinhibited in the patient.

Also provided by the present invention is a combination therapeuticagent. The combination agent comprises an inhibitor of COX2 and aprotein binding inhibitory agent selected from the group consisting of:an antibody which binds to amino acid residues 1-114 of human iNOS oramino acid residues 484-604 of human COX2; a polypeptide comprising afragment of human iNOS from the N-terminal 10% of iNOS sufficient tobind COX2 or a fragment of human COX2 from the C-terminal 20% of COX2sufficient to bind iNOS; or a nucleic acid vector encoding a polypeptidecomprising a fragment of human iNOS from the N-terminal 10% of iNOSsufficient to bind COX2 or a fragment of human COX2 from the C-terminal20% of COX2 sufficient to bind iNOS.

These and other embodiments which will be apparent to those of skill inthe art upon reading the specification provide the art with methods ofidentifying potential anti-inflammatory agents and with therapeuticanti-inflammatory agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1H. COX2 and iNOS bind selectively in vitro and in intact cells.(FIG. 1A) RAW264.7 cells were induced with LPS(2 μg/ml)/IFNγ(100 U/ml).COX2 was immunoprecipitated by anti-COX2 antibodies and probed usinganti-COX and anti-iNOS antibodies. (FIG. 1B and FIG. 1C) RAW264.7 cellswere induced by LPS/IFNγ with or without an iNOS inhibitor 1400W (100μM) or COX2 inhibitor SC58125 (100 μM). Cell lysates were subjected toimmunoprecipitation with anti-COX2 and anti-iNOS antibodies. (FIG. 1D)The fragments of iNOS denoted in red bound to COX2, while fragmentslabeled purple did not as determined by co-immunoprecipitation. (FIG.1E) HEK293T cells transfected with COX2 and GST-fused iNOS fragmentswere immunoprecipitated with GST conjugated beads. Proteins weredetected by the addition of GST-HRP and goat anti-COX2 antibodies. (FIG.1F) HEK293T cells transfected with COX2 and myc-tagged iNOS fragmentswere immunoprecipitated with anti-myc antibodies. (FIG. 1G) Generatedfragments of COX2 which bind to full length iNOS are labeled in red,while those that do not bind are identified in yellow. (FIG. 1H) HEK293Tcells transfected with iNOS and myc-tagged COX2 fragments wereimmunoprecipitated by anti-myc.

FIG. 2A-2G. S-nitrosylation of COX2 enhances enzyme activity. (FIG. 2A)COX2 expressed in HEK293T cells is S-nitrosylated in the presence ofGSNO (100 μM) as determined by biotin switch assay. (FIG. 2B) LPS/IFNγtreatment of RAW264.7 cells elicits S-nitrosylation of COX2 which issubsequently prevented by the iNOS inhibitor 1400W (100 μM). (FIG. 2C)COX2 enzyme activity was measured from the cell lysate of COX2transfected HEK293T cells in the presence or absence of SNP (200 μM) andascorbate (ASC, 500 μM). Bars represent the mean±SE of three independentcell cultures performed in triplicate. (* Student's t-test) (FIG. 2D)Transfected COX2-myc is S-nitrosylated in the presence of SNP (200 μM)and reversed by the addition of 500 μM ASC. (FIG. 2E) COX2-myc expressedin HEK293T cells is S-nitrosylated by various concentration of GSNO. Thedose-dependence of GSNO-mediated activation of PGE2 (ng/mL) wasmeasured. Data were pooled from at least three independentdeterminations each in triplicate. (FIG. 2F) Recombinant human COX2 wastreated with SNP (1 mM) and COX2 activities were measured. (n=3, •=CTL,Vmax=81.3±4.8 nmol/min/mg, Km=16.2±2.2 μM; o=1 mM SNP, Vmax=132±6.5nmol/min/mg, Km=17.0±2.0 μM) (FIG. 2G) Recombinant human COX2 wastreated with SNP (1 mM) and its turnover rate (kcat) was measured in thepresence of various concentration of sucrose. Data was expressed asKcat-control over Kcat in each viscosity vs. viscosity ratio.

FIG. 3A-3D. Endogenously generated NO enhances COX2 activity. (FIG. 3A)RAW264.7 cells were activated by LPS/IFNγ at various concentrations of1400W overnight. The dose dependence of 1400W mediated suppression ofPGE2 (ng/mL) and nitrite (μM) levels were then measured. Data werepooled from at least three independent determinations each intriplicate. (* Student's t-test) (FIG. 3B) Combinations of L-NAME (500μM), L-NAME+L-Arg (1 mM) or D-Arg (1 mM), and D-NAME (500 μM) were addedto RAW264.7 cells subjected to LPS/IFNγ. PGE2 levels were measured andthe data are pooled from three independent experiments performed each intriplicate. (FIG. 3C) PGE2 and nitrite levels were measured from wildtype and iNOS knock-out primary peritoneal macrophages treated with orwithout LPS/IFNγ. (* Student's t-test) (FIG. 3D) S-nitrosylation of COX2of wild type primary peritoneal macrophages treated with LPS/IFNγ, isabolished in iNOS knock-out cultures.

FIG. 4A-4D. COX2-myc fragment attenuates iNOS binding to COX2 andNO-mediated activation of PGE2 production. RAW264.7 cells transfectedwith COX2-myc fragments 1-483 and 484-604 were treated with LPS/IFNγ.(FIG. 4A) Cell lysates were immunoprecipitated with rabbit anti-iNOSantibodies and analyzed with mouse anti-iNOS, goat anti-COX2 and mouseanti-myc antibodies. (FIG. 4B) COX2-myc fragment (484-604) decreasesS-nitrosylation of COX2 in RAW264.7 cells. (FIG. 4C) RAW264.7 cells weretransfected with the fragments and treated with LPS/IFNγ. PGE2 levelswere measured and the data are pooled from three independent experimentsperformed each in triplicate. (* Student's t-test) (FIG. 4D). PGE2 andCOX2 fragments were visualized with confocal microscopy using mouseanti-myc and Rabbit anti-PGE2 antibodies. Images of COX2 (red) and PGE2(green) were superimposed to show co-localization. Nuclei werevisualized using Hoechst staining (blue). In FIG. 4D1 arrows point totwo RAW264.7 cells, only one of which is transfected with the COX2fragment 484-604 (red). In FIG. 4D2, the same two cells are analyzed forpresence of endogenous PGE2 produced after activation of RAW264.7 cellsby LPS/IFNγ treatment. Immunofluorescent staining shows a reduction inthe PGE2 expression level in the COX2-myc 484-604 transfected cellcompared to the non-transfected cell (FIG. 4D2). This observationcontrasts with FIG. 4D4 whose arrows point to a transfected andnon-transfected cell of the COX2 fragment 1-483. Unlike FIG. 4D2, FIG.4D5 does not show a reduction of PGE2 in the transfected cell ascompared to the non-transfected cell.

FIG. 5A-5B. (S1) COX2 and iNOS bind in vivo. (FIG. 5A) HEK293T cellstransfected with COX2, and iNOS were subjected to immunoprecipitationwith anti-myc antibody and analyzed with mouse anti-iNOS and goatanti-COX2 antibodies. (FIG. 5B) Peritoneal macrophages were obtainedeither 4 or 22 hours after 3% thioglycolate injection. Cell lystes wereimmunoprecipitated by rabbit anti-iNOS antibody and analyzed with ratanti-COX2 and mouse anti-iNOS antibodies.

FIG. 6A-6B (S2.) GST-tagged iNOS fragments co-immunoprecipitate withCOX2 in vitro. (FIG. 6A) GST-tagged iNOS fragments for amino acidresidues 1-500 and 1-525 were respectively combined with full lengthCOX2 and pulled down using GST beads. Purified GST was also combinedwith COX2 as a control. (FIG. 6B) GST-tagged iNOS fragments for aminoacid residues 1-500 and 510-1145 were co-immunoprecipitated with COX2using GST pull down. These results suggest that interaction between iNOSand COX2 is direct binding.

FIG. 7A-7B (S3.) COX2 is S-nitrosylated by NO in vitro and in vivo.(FIG. 7A) COX2 is selectively S-nitrosylated in the presence ofadditional NO donors DETA-NO (500 μM), SNP (100 μM), and Spermine-NO(100 μM) using the biotin switch assay. (FIG. 7B) S-nitrosylation ofCOX2 in RAW264.7 cells treated with LPS/IFNγ was S-nitrosylated usingfluorometic detection assay, which was abolished by iNOS specificinhibitor, 1400W.

FIG. 8A-8B (S4.) Biotin switch assay is specific for S-nitrosothioldetection. (FIG. 8A) COX2 transfected in HEK293T cells were treated witheither GSNO or H2O2 to generate S-nitrosothiol or sulfenic acid,respectively. It has been known that 10 mM arsenite is a reducingreagent specific for sulfenic acid. Biotin switch assay was performedwith either 1 mM ascorbate or 10 mM sodium arsenite. Our results showthat ascorbate is specific for S-nitrosylated COX2 not for COX2 withsulfenic modification, which was confirmed by reversing it using sodiumarsenite. (FIG. 8B) S-nitrosylation of COX2 in RAW264.7 cells treatedwith LPS/IFNγ was confirmed using biotin switch assay either withascorbate or sodium arsenite. Ascorbate treatment reversed COX2modification in RAW264.7 cells treated with LPS/IFNγ but sodium arsenitedid not, showing that COX2 in LPS/IFNγ-treated RAW264.7 cells is mostlyS-nitrosylated.

FIG. 9A-9B. (S5.) Oxyhemoglobin blocks S-nitrosylation of COX2 byexogenous NO but not by endogenous NO produced by iNOS in activatedRAW264.7 cells. (FIG. 9A) HEK293T cells transfected with COX2-myc weretreated with 100 μM GSNO for 3 hours with or without oxyhemoglobin andbiotin switch assay was performed. Oxyhemoglobin blocked GSNO-mediatedS-nitrosylation of COX2. (FIG. 9B) However, oxyhomoglobin did notprevent S-nitrosylation of COX2 in RAW264.7 cells induced by LPS/IFNγsuggesting that a delivery of NO to COX2 is crucial.

FIG. 10A-10B (S6.) Target of S-nitrosylation of COX2 is located inc-terminus (484-604) and mutation of Cysteine526 to Serine preventsNO-mediated activation of COX2. (FIG. 10A) COX2 has 13 cysteines and wemutated all the cysteines to serine, two of which were not expressed.Single mutation of cysteine to serine did not eliminate S-nitrosylationsignal by the biotin switch assay (data shown) suggesting that there aremore than one cysteine can be S-nitrosylated. Hence, we performed biotinswitch assay using COX2 fragments and demonstrated that the target forS-nitrosylation is located in c-terminal region (484-604). (FIG. 10B) Wewondered which of the 13 cysteines of COX2 are critical for theS-nitrosylation elicited augmentation of COX2 activity. COS1 cells weretransfected with empty vector, wild type, or mutants of COX2-myc. Cellswere treated with (red) or without (black) NO donor for 3 h, at whichpoint PGE₂ levels were measured. Mutation of C526 to serine (C526S)abolishes stimulation of PGE2 formation by the NO donor SNP, while C561Sfails to influence stimulation. The target cysteine which is responsiblefor NO-mediated COX2 activation is also located in the region of484-604. We attempted to study COX2 with mutation of the other 11cysteines, two of which could not be expressed in COS1 cells while theother 9 mutations had no demonstrable catalytic activity even undercontrol conditions (data not shown). We could not eliminate thepossibility that there is more than one S-nitrosylation target butidentified the target cysteine responsible for NO-mediated activation ofCOX2.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have found that iNOS binds directly to COX2 without ascaffold protein. Moreover, we found that iNOS binding to COX2 leads toS-nitrosylation and activation of COX2. We further found that thebinding of iNOS to COX2 leads to augmented formation of prostaglandinsby COX2. Based on these findings, one can screen and develop novelanti-inflammatory drugs by monitoring the ability of candidate drugs toinhibit the binding of iNOS to COX2. Peptide agents that mimic theportions of the two binding partners that interact with each other, canbe used to block the activation of COX2 by iNOS. Similarly antibodieswhich bind to the interacting portions of the binding partners can beuse to inhibit inflammation responses.

For screening assays, any molecules comprising the binding domains ofCOX2 and iNOS can be used. These include full-length COX2 and iNOSpolypeptides, fragments comprising the binding domains of the twofull-length polypeptides, fusion proteins comprising the binding domainsof the two full length polypeptides. As defined experimentally, thebinding domains are within the N-terminal 10% of iNOS and the C-terminal20% of COX2, as these residues have been found to be sufficient tomediate binding. Fragments contain less than the full-length proteins,typically less than 50%, more typically less than 25%. Fragments of COX2do not contain the membrane binding domain while fragments of iNOS donot contain the reductase or CaM (calmodulin) domains. Any of theseforms of iNOS protein can be used to bind to any form of the COX2protein. Binding can be determined by measuring one or both of thebinding partners, either in the bound or the free form. Increase in thebound form or decrease in the free form indicates binding of the twopartners. Conversely, added test compounds which inhibit binding can becharacterized by the decrease in bound form or increase in free form ofone or both of the binding partners.

For screening assays, the test compound is preferably a small moleculethat binds to and occupies, for example, the binding site of the COX2 oriNOS polypeptide, such that normal binding of the two binding partnersis prevented. Examples of such small molecules include, but are notlimited to, small peptides or peptide-like molecules. Any small organicmolecule can be used. Libraries of natural or synthetic compounds can bescreened. Small molecules which mimic three dimensional structure ofpeptides can be designed.

In screening assays, either the COX2 or iNOS polypeptide can comprise adetectable label, such as a fluorescent, radioisotopic,chemiluminescent, or enzymatic label, such as horseradish peroxidase,alkaline phosphatase, or luciferase. Detection of bound COX2 or iNOSpolypeptide can then be accomplished, for example, by direct counting ofradioemmission, by scintillation counting, or by determining conversionof an appropriate substrate to a detectable product.

Alternatively, inhibition of binding of a human COX2 to an iNOSpolypeptide by a test compound can be determined without labeling theinteractants. For example, a microphysiometer can be used to detectbinding of a human COX2 and iNOS polypeptide. A microphysiometer (e.g.,Cytosensor™) is an analytical instrument that measures the rate at whicha cell acidifies its environment using a light-addressablepotentiometric sensor (LAPS). Changes in this acidification rate can beused as an indicator of the interaction between a human COX2 or iNOSpolypeptide (McConnell et al., Science 257, 1906-1912, 1992).

Determining the ability of a mammalian COX2 and an iNOS polypeptide tobind also can be accomplished using a technology such as real-timeBimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal.Chem. 63, 2338-2345, 1991, and Szabo et al., Curr. Opin. Struct. Biol 5,699-705, 1995). BIA is a technology for studying biospecificinteractions in real time, without labeling any of the interactants(e.g., BIAcore™). Changes in the optical phenomenon surface plasmonresonance (SPR) can be used as an indication of real-time reactionsbetween biological molecules.

In yet another aspect of the invention, a mammalian COX2 or iNOSpolypeptide can be used in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., Cell 72, 223-232,1993; Madura et al., J. Biol. Chem. 268, 12046-12054, 1993; Bartel etal., BioTechniques 14, 920-924, 1993; Twabuchi et al., Oncogene 8,1693-1696, 1993; and Brent WO94/10300), to identify agents which inhibitbinding of the COX2 and iNOS polypeptides and thereby modulate COX2activity.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. For example, in one construct, polynucleotide encoding amammalian COX2 or iNOS polypeptide can be fused to a polynucleotideencoding the DNA binding domain of a known transcription factor (e.g.,GAL-4, particularly yeast GAL-4). In the other construct a DNA sequencethat encodes the other of COX2 or iNOS can be fused to a polynucleotidethat codes for the activation domain of the known transcription factor.When the fusion proteins are able to interact in vivo to form aprotein-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ, particularly E.coli LacZ), which is operably linked to a transcriptional regulatorysite responsive to the transcription factor. Contacting such cells(typically but not limited to yeast cells) with test substances willpermit an assay for binding inhibition. Expression or inhibition ofexpression of the reporter gene can be detected, thus identifying anagent which inhibits the interaction of COX2 and iNOS polypeptide.

It may be desirable to immobilize either the COX2 or iNOS polypeptide orthe test compound to facilitate separation of bound from unbound formsof one or both of the interactants, as well as to accommodate automationof the assay. Thus, either the COX2 or iNOS polypeptide or the testcompound can be bound to a solid support. Suitable solid supportsinclude, but are not limited to, glass or plastic slides, tissue cultureplates, microtiter wells, tubes, silicon chips, or particles such asbeads (including, but not limited to, latex, polystyrene, or glassbeads). Any method known in the art can be used to attach thepolypeptide or test compound to a solid support, including use ofcovalent and non-covalent linkages, passive absorption, or pairs ofbinding moieties attached respectively to the polypeptide or testcompound and the solid support. Test compounds can be bound to the solidsupport in an array, so that the location of individual test compoundscan be tracked. Binding of a test compound to a mammalian COX2 or iNOSpolypeptide can be accomplished in any vessel suitable for containingthe reactants. Examples of such vessels include microtiter plates, testtubes, and microcentrifuge tubes.

In one embodiment, the iNOS polypeptide is a fusion protein comprising adomain that allows the iNOS polypeptide to be bound to a solid support.For example, glutathione-S-transferase fusion proteins can be adsorbedonto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and the non-adsorbed COX2polypeptide; the mixture is then incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads or microtiter plate wells are washed toremove any unbound components. Binding of the interactants can bedetermined either directly or indirectly, as described above.Alternatively, the complexes can be dissociated from the solid supportbefore binding is determined. In another alternative, the COX2polypeptide is a fusion protein.

Other techniques for immobilizing proteins on a solid support also canbe used in the screening assays of the invention. For example, either aniNOS or COX2 polypeptide or a test compound can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated iNOS or COX2polypeptides test compounds can be prepared frombiotin-NHS(N-hydroxysuccinimide) using techniques well known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.) andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies which specifically bind to an iNOSor COX2 polypeptide, or a test compound, but which do not interfere witha desired binding site, can be derivatized to the wells of the plate.Unbound target or protein can be trapped in the wells by antibodyconjugation.

Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies which specifically bind to the iNOS or COX2polypeptide or test compound, enzyme-linked assays which rely ondetecting an activity of the iNOS or COX2 polypeptide, and SDS gelelectrophoresis under non-reducing conditions.

Screening for test compounds which bind to an iNOS or COX2 polypeptidealso can be carried out in an intact cell, in whole animals, orcell-free systems. Any cell which comprises an iNOS or COX2 polypeptidecan be used in a cell-based assay system. An iNOS or COX2 polynucleotidecan be naturally occurring in the cell or can be introduced usingtechniques such as those described above. Binding of the test compoundto an iNOS or COX2 polypeptide is determined as described above.

Once a test compound has been identified that inhibits the binding ofiNOS to COX2, whether in cells, in whole animals, or in a cell-freesystem, it can be further tested to determine its effect oninflammation. For example, it can be tested for its effect onprostaglandin synthesis or for its effect in any of several animalmodels of inflammation known in the art. Exemplary animal models includethe following mouse strains, without limitation. The mouse strainCD1-Tg(Gadd45β-luc)-Xen (Gadd45β-luc LPTA®) animal model is useful instudying apoptosis, Gadd45β gene regulation, MAP kinase- andNF-κB-mediated signaling pathways, and the treatment of inflammatorydiseases and cancer. The mouse strain FVB/N-Tg(iNos-luc)Xen (iNos-lucLPTAR®) animal model is useful in studying sepsis, arthritis, andanti-inflammatory compounds. The mouse strain FVB/N-Tg(Epx-luc)-Xen(Epx-luc LPTA®) animal model is useful in studying changes in eosinophilproduction resulting from parasite infection and asthma, and may be usedas donor animals for studying bone marrow transplantation. The mousestrain BALB/C-Tg(Saa1-luc)-Xen (Saa1-luc LPTA®) animal model is usefulin studying sepsis, arthritis, amyloidosis, and A-SAA-mediateddisorders. The mouse strain CD1-Tg(IL2-luc)-Xen (IL2-luc LPTA®) animalmodel is useful in studying IL-2 gene regulation, inflammatory diseases,and cancer. The mouse strain BALB/C-Tg(Tnfα-luc)-Xen (Tnfα-luc LPTA®)animal model is useful in studying sepsis, arthritis, inflammatory boweldisease, apoptosis, TNFα gene regulation, and the treatment ofTNFα-mediated inflammatory diseases. The mouse strainBALB/C-Tg(NFκB-RE-luc)-Xen (NFκB-RE-luc LPTA®) animal model is useful instudying sepsis, arthritis, inflammatory bowel disease, apoptosis, tumordevelopment, NFκB gene regulation, and the treatment of inflammatorydiseases and cancer. The mouse strain DBA/1, BALB/C-Tg (NFkB-RE-luc[Oslo])-Xen (NFκB-luc (Oslo) LPTA®) animal model is useful in studyingsepsis, arthritis, inflammatory bowel disease, apoptosis, tumordevelopment, NFκB gene regulation, and the treatment of inflammatorydiseases and cancer. The mouse strain BALB/C-Tg(IkBa-luc)-Xen (IκBa-lucLPTA®) animal model is useful in studying sepsis, arthritis,inflammatory bowel disease, apoptosis, tumor development,transcriptional regulation of IκBα gene and other genes responsive toNFκB, and the treatment of inflammatory diseases and cancer. Any animalmodel of inflammation can be used.

COX2 and iNOS for use in any of the methods or compositions of theinvention can be from any species, particularly from mammals, such asrat, mouse, rabbit, dog, cat, and most particularly from humans. Theseproteins are well known in the art and a version of these proteins fromany individual can be used even though the version of the protein fromthat individual may vary slightly from that which is documented in aprotein sequence database. Exemplary sequences which can be used arethose which are documented at GenBank as P35228 and P35354. Other COX2sequences which can be used include those from P. troglodytes, C.familiaris, M. musculus, R. norvegicus, and G. gallus. Exemplarysequences include XP-520238, NP_(—)001003023, NP_(—)032995,XP_(—)579388, and XP_(—)425326, respectively. Other iNOS sequences whichcan be used include those from C. familiaris, M. musculus, R.norvegicus, and G. gallus. Exemplary sequences include NP_(—)001003186,NP_(—)035057, and NP_(—)990292, respectively. All referenced proteinand/or gene sequences are incorporated by reference herein as they existon Apr. 26, 2006.

Treatment modalities based on the discovery of binding of iNOS and COX2include administration of a polypeptide, administration of a nucleicacid encoding a polypeptide, and administration of an antibody. Thenucleic acid can be a naturally occurring genomic or cDNA sequence orcan be any sequence which encodes the desired protein. Each of these isdesigned to inhibit binding of the two binding partners. Both thepolypeptides and the antibodies bind to the portion of the bindingpartners that interact, thereby inhibiting the interaction. Inhibitionof the interaction inhibits the iNOS-mediated S-nitrosylation of COX2which activates COX2. Polypeptides delivered directly or indirectly vianucleic acids can be fragments of fusion proteins comprising a bindingdomain or sufficient amounts of a binding domain to bind and interferenwith COX2/iNOS binding.

Inflammation which can be treated according to the present inventionincludes any which is exacerbated by prostaglandin synthesis. Theseinclude, without limitation, chronic inflammatory diseases, such asrheumatoid arthritis, inflammatory bowel disease, systemic lupuserythematosus, multiple sclerosis, type Idiabetes, rheumatoid arthritis,chronic obstructive pulmonary disease (COPD), asthma, allergic rhinitis,cardiovascular disease, psoriasis. Inflammatory diseases of the braininclude abscess, meningitis, encephalitis and vasculitis. Any of thesediseases can be treated according to the methods of the presentinvention.

Antibodies for use in the present invention can be monoclonal orpolyclonal. They can gain their specificity by purification or bylimitation of the inducing immunogen. The antibodies will bind to theN-terminal 10% of iNOS or to the C-terminal 20% of COX2. Administrationof antibodies can be by any means known in the art, but typically theantibodies are administered by injection, such as intrathecal,intraventricular, intravascular (intravenous or intraarterial),subcutaneous, intramuscular, intraperitoneal, intrapleural, by perfusionthrough a regional catheter, or by direct intralesional injection. Whenadministering antibodies by injection, the administration may be bycontinuous infusion or by single or multiple boluses. Polypeptides andpolynucleotides can be administered by similar means.

In general, the dosage of administered antibodies will vary dependingupon such factors as the patient's age, weight, height, sex, generalmedical condition, and previous medical history. Preferably, asaturating dose of antibody is administered to a patient. Antibodies canbe administered as whole IgG, F(ab′)₂, F(ab)₂, Fab′, or Fab.

Typically, it is desirable to provide the recipient with a dosage ofantibody that is in the range of from about 50 to 500 milligrams ofantibody, although a lower or higher dosage also may be administered ascircumstances dictate. Effective in vivo dosages of an antibody are inthe range of about 5 mg to about 50 mg/kg, about 50 mg to about 5 mg/kg,about 100 mg to about 500 mg/kg of patient body weight, and about 200 toabout 250 mg/kg of patient body weight. High doses of antibody may causeanaphylaxis due to complement activation with endogenous antibodies.This side effect, however, can be prevented by administration ofoligosaccharides that bind with endogenous antibodies, as detailedbelow.

The antibodies of the present invention can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebyantibodies are combined in a mixture with a pharmaceutically acceptablecarrier. A composition is said to be a “pharmaceutically acceptablecarrier” if its administration can be tolerated by a recipient patient.Sterile phosphate-buffered saline is one example of a pharmaceuticallyacceptable carrier. Other suitable carriers are well-known to those inthe art. See, for example, Remington's Pharmaceutical Sciences, 19th Ed.(Mack Publishing Co. 1995), and Gilman's The Pharmacological Basis ofTherapeutics, 7th Ed. (MacMillan Publishing Co. 1985).

For purposes of therapy, an antibody and a pharmaceutically acceptablecarrier are administered to a patient in a therapeutically effectiveamount. A combination of an antibody and a pharmaceutically acceptablecarrier is said to be administered in a “therapeutically effectiveamount” if the amount administered is physiologically significant. Anagent is physiologically significant if its presence results in adetectable change in the physiology of a recipient patient. In thepresent context, an agent is physiologically significant if its presenceresults in the modulation of an immune response or malignant T cellmalignancy growth.

Polynucleotides can be administered in any vector, whether viral ornon-viral designed for delivery and expression of inserted nucleic acidsequences. Polynucleotides and/or proteins can be further formulated inliposomes or cationic vesicles or particles for added stability. Viralvectors include adenovirus vectors, adeno-associated virus vectors,retrovirus vectors, lentivirus vectors. Non-viral vectors includeplasmid vectors. Exemplary types of viruses include HSV (herpes simplexvirus), AAV (adeno associated virus), HIV (human immunodeficiencyvirus), BIV (bovine immunodeficiency virus), and MLV (murine leukemiavirus). Nucleic acids can be administered in any desired format thatprovides sufficiently efficient delivery levels, including in virusparticles, in liposomes, in nanoparticles, and complexed to polymers.

Inducible NOS and COX2 physiologically bind, bringing NO in proximitywith COX2, facilitating its S-nitrosylation and activation. Earlierfindings that NOS inhibition decreases prostaglandin formation suggesteda relationship between the two systems(10, 26), but might have reflecteda generally diminished stressful stimulus to the COX2 system rather thana direct intermolecular linkage. Because NO is a labile molecule whichcan be rapidly inactivated within cells by the high physiologicconcentrations of glutathione or other reducing agents, it is possiblethat many of NO's physiologic actions will require delivery of NO to itstargets(15). Other instances of NO delivery have primarily involved nNOSacting via scaffold proteins. Thus, a scaffold protein CAPON links nNOSto Dexrasl and provides NO to S-nitrosylate Dexrasl and act as itsguanine nucleotide exchange factor(27). Similarly the scaffold proteinPSD95 links nNOS to N-Methyl-D-aspartate (NMDA) receptors where NOS-nitrosylates and inactivates NMDA receptors(28). In contrast to theseexamples, iNOS binds directly to COX2 with no intervening scaffoldprotein. A similar direct delivery of a regulatory metabolite viaprotein-protein interactions involves the binding ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH) to inositol1,4,5-trisphosphate (IP3) receptors with NADH formed by GAPDHselectively augmenting calcium release activity of IP3 receptors(29).

The molecular synergism between iNOS and COX2 may represent a majormechanism of inflammatory responses. Inhibitors of iNOS do relieve feverand pain, classically associated with prostaglandin production which mayreflect the iNOS-COX2 interaction, though such actions are sufficientlyindirect that one cannot draw strong conclusions(30, 31).

Our findings have therapeutic relevance. Thus drugs which block theiNOS-COX2 interaction might have anti-inflammatory action. Moreover,such agents might synergize with COX2 inhibitor drugs permitting lowerdoses with less side-effects. While it has been speculated that adversecardiovascular effects of COX2 inhibitors reflect inhibition of PGE₂formation, this has not been directly established so that other actionsof the drugs might be involved(32).

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

EXAMPLE 1 Materials and Methods Materials and Methods

Cells. HEK293T, COS1 and RAW 264.7 murine macrophages were obtained fromthe American Type Culture Collection (Manassas, Va.). They were grown ina humid atmosphere of 95% air and 5% CO2 at 37° C. in Dulbecco'smodified Eagle's medium supplemented with 10% fetal bovine serum,L-glutamine (300 ug/ml), penicillin (100 U/ml), and streptomycin (100μg/ml).

Preparation of peritoneal macrophages. Peritoneal macrophages wereobtained following intraperitoneal injection of 1.5 ml of 3% sterilethioglycolate medium. After 4 days mice were sacrificed and macrophageswere harvested as described(1).

Plasmid Constructions. Full length human cyclooxygenase2-encoding gene(gene bank sequence NM_(—)000963) was purchased from ATCC in pCMV-SPORT6vector. The sequence was amplified by PCR using primers harboringSal1/Not1 restriction sites and cloned into the pCMV-Myc vector(Clontech, Palo Alto, Calif.). The murine inducible nitric oxidesynthase (accession number NM_(—)010927) cloned into the pcDNA3.1+vector(Invitrogen, Carlsbad, Calif.).

Immunoprecipitation. For co-immunoprecipitation experiments, 1×10⁶HEK293T cells were plated in 10 cm² culture dishes (Invitrogen,Carlsbad, Calif.). Cells were transfected with 4 μg pCMV-Myc-COX2, 2 ugpcDNA3.1-iNOS, or empty pCMV-Myc vector using LipofectAMINE PLUS(Invitrogen, Carlsbad, Calif.) according to the manufacturer's protocol.After 48 h, cells were harvested and lysed in ice-cold lysis buffer (100mM Tris pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, and completeprotease inhibitors). The supernatants (800 ul) from the HEKT cellextracts were pre-cleaned for non-specific binding with 50 ul proteinA-Sepharose, then mixed overnight at 4° C. with 2 ug/ml anti-Mycantibody (Roche, Alameda, Calif.). After the addition of 80 μl ofprotein A-Sepharose, the immunoprecipitates were mixed for another 1 hat 4° C. The mixture was washed three times with the buffer describedabove and the pellet boiled in 10 ul SDS-loading dye, which wassubjected to 6% SDS PAGE gel run in MOPS buffer. The proteins were thentransferred to nitrocellulose membrane. The bands were visualized by ECLreagent (Pierce, Milwaukee, Wis.) as described by the manufacturer.

Site Directed Mutagenesis. The QuickChange site-directed mutagenesissystem (Stratagene, La Jolla, Calif.) was employed per manufacturer'sinstructions to alter the thirteen cysteine residues in COX2 to serine.Each mutant was verified via automated sequencing by the Hopkins CoreFacility.

S-nitrosylation assay. Cells were homogenized by 26G needle in HEN (250mM Hepes-NaOH pH 7.7, 1 mM EDTA, 0.1 mM Neocuproine) buffer and thencentrifuged at 1000 g for 10 min at 4° C. Cells lysates (240 μg) wasadded 4 vol of blocking buffer (9 vol of HEN buffer plus 1 vol 25% SDS,adjusted to 20 mM MMTS with a 2 M stock prepared in dimethylformamide(DMF)) at 50° C. for 20 min with frequent vortexing. The MMTS was thenremoved by desalting three times with the MicroBioSpin6 column (Bio-Rad,Hercules, Calif.) pre-equilibrated in HEN buffer. To the eluate wasadded biotin-HPDP prepared fresh as a 4 mM stock in DMSO from a 50 mMstock suspension in DMF. Sodium ascorbate was added to a finalconcentration of 1 mM. After incubation for 1 h at 25° C., biotinylatedproteins were precipitated by streptavidin-agarose beads. Thestreptavidin-agarose was then pelleted and washed 5 times using HENSbuffer. The biotinylated proteins were eluted by SDS-PAGE sample bufferand subjected to Western-blot analysis.

Fluorometric detection of S-nitrosothiols. The methods were modifiedaccording to Cook et al. (2) RAW264.7 cells were treated with LPS/IFNγfor overnight and COX2 was immunoprecipitated by donkey anti-rat COX2antibodies. After that the samples were reacted with 100 μM2,3-diaminonaphthalene (DAN) in the presence of 100 μM of HgCl2 andincubated in darkness for 30 min at room temperature. The generatedfluorescent compound 2,3-napththyltrazole (NAT) was then measured at anexcitation wavelength of 375 nm and an emission wavelength of 450 nm.

COX2 enzymatic assay. Recombinant human COX2 was obtained from CaymanBiochemical Inc. COX2 was treated with NO donor, SNP for 30 min and wasthen passed through a spin column (Bio-Rad, Hercules, Calif.) to removeexcess NO. COX2 enzymatic activity was measured with a COX assay kit(Cayman Biochemical Inc, Ann Arbor, Mich.) according to themanufacturer's instructions.

Measurement of PGE₂. Cells were washed with worm PBS (x2) and incubatedin the phenol free Dulbecco's modified Eagle's medium supplemented with20 μM arachidonic acid and 3% albumin for 20 min. Media was collectedand PGE2 production was measured with a PGE₂ ELISA kit from Assay Design(Ann Arbor, Mich.) according to the manufacturer's instructions.

Immunohistochemistry. RAW264.7 cells were transfected with deletionconstructs of COX2. Cells were fixed in 4% paraformaldehyde in PBS for 5min, permeablized in 0.1% Triton X-100 for 10 s, and then rinsed twicein PBS. Coverslips were then blocked in 10% goat serum at roomtemperature for 1 h and incubated with PGE₂ (Cayman Biochemical Inc, AnnArbor, Mich.) and myc antibodies for 24 hr at 4° C. Rhodamine orfluorescein-conjugated secondary antibodies (Jackson Immunochemicals,West Grove, Pa.) were then added at 10 μg/ml for 1 hr at roomtemperature as indicated. Coverslips were mounted in ProLong (MolecularProbes, Eugene, Oreg.). Confocal microscopy, in which theimmunofluorescent staining is superimposed on phase contrast images,employed a Noran OZ (Noran Instruments, Middleton, Wis.) confocallaser-scanning system, fitted to an Olympus IX-50 fluorescencemicroscope.

Viscosity studies. Viscosity experiments were performed with sucrose(0-31% w/w) as described previously (3, 4).

1. Nunoshiba, T., deRojas-Walker, T., Wishnok, J. S., Tannenbaum, S. R.& Demple, B. Activation by nitric oxide of an oxidative-stress responsethat defends Escherichia coli against activated macrophages. Proc NatlAcad Sci USA 90, 9993-7 (1993).

2. Cook, J. A., Kim, S. Y., Teague, D., Krishna, M. C., Pacelli, R.,Mitchell, J. B., Vodovotz, Y., Nims, R. W., Christodoulou, D., Miles, A.M., Grisham, M. B., & Wink, D. A. Convenient colorimetric andfluorometric assays for S-nitrosothiols. Anal Biochem 238, 150-158(1996).

3. Cole, P. A., Burn, P., Takacs, B., Walsh, C. T. Evaluation of thecatalytic mechanism of recombinant human Csk (C-terminal Src kinase)using nucleotide analogs and viscosity effects. J Bio Chem 269,30880-30887 (1994).

4. Brouwer, A. C., and Kirsch, J. F. Investigation of diffusion-limitedrates of chymotrypsin reactions by viscosity variation. Biochem 21,1302-1307 (1982)

EXAMPLE 2 iNOS and COX2 Bind in Vivo in Cells and Whole Animals

For initial studies we employed RAW264.7 cells, a macrophage cell linein which LPS and IFNγ massively activate both iNOS and COX2. In LPS/IFNγtreated cells immunoprecipitation with COX2 antibodies leads toco-precipitation of iNOS (FIG. 1A). Coprecipitation of COX2 and iNOS isalso evident in HEK293T cells transfected with both proteins (FIG. 5A).

To examine interactions between the two enzymes in intact organisms, weinjected mice with thioglycollate, an inflammatory stimulus whichtypically produces peritonitis or pleuritis, and observedcoprecipitation of iNOS and COX2 (FIG. 5B).

To determine whether catalytic activity of the enzymes influences theirbinding interactions, we co-precipitated the two proteins with iNOSantibodies or COX2 antibodies and examined the effect of theiNOS-selective inhibitor 1400W (FIG. 1B) or the COX2 selective inhibitorSC58125 (FIG. 1C). Co-precipitation of iNOS and COX2 is unaffected byeither iNOS or COX2 inhibitors. The binding of iNOS and COX2 isselective, as we do not detect immunoprecipitation of COX1 with iNOS(data not shown).

EXAMPLE 3 Mapping the Binding Sites on the Two Interacting Proteins

To map the binding sites, we employed selective deletions of iNOS (FIG.1D-F) and COX2 (FIG. 1G and H) sequences. Amino acids 1-114 appear to bethe minimal portion of iNOS mediating binding, while amino acids 484-604of COX2 are required. The binding area of iNOS occurs within theoxygenase domain, while the C-terminal portion of COX2 that mediatesbinding includes a COX2 domain which does not exist in COX1.

EXAMPLE 4 NO Donors Elicit S-Nitrosylation of COX2

The two major mechanisms whereby NO influences its targets arestimulation of guanylyl cyclase by direct binding of NO to iron in hemeat the active site of guanylyl cyclase(11, 12) or S-nitrosylation ofprotein targets on appropriate cysteines(13-15). Since COX2 possessesheme at its active site(16), this would be a potential target. However,NO binding to heme in COX does not alter its activity(17).

COX2 also contains 13 cysteines whose roles are not fully understood(18,19). Hence, we explored the possibility of S-nitrosylation of COX-2 byNO, examining multiple NO donors including nitrosoglutathione (GSNO)(FIG. 2A), sodium nitroprusside (SNP), spermine-NO and DETA-NONOate(FIG. 7A). Utilizing the biotin switch method(20), we demonstrate thatall four NO donors elicit S-nitrosylation of COX2 in HEK293T cellstransfected with COX2-myc (FIG. 2A).

We wondered whether physiological induction of NO formation leads toS-nitrosylation of COX2. In RAW264.7 cells treated with LPS/IFNγ weobserve S-nitrosylation of COX2 which is prevented by the iNOS specificinhibitor 1400W using the biotin switch assay (FIG. 2B) as well as thefluorometric method (FIG. 7B). To ensure specificity of the biotinswitch method, we have observed that H₂O₂ does not elicitS-nitrosylation (FIG. 8A). We ruled out the possibility that sulfenicacid modification is detected by the biotin switch assay bydemonstrating that arsenite, which reverses sulfenic acid modificationsbut not S-nitrosylation, fails to provide the biotin switch signalafforded by ascorbate utilizing GSNO with purified COX2 or LPS/IFNγtreatment of RAW 264.7 cells (FIG. 8B).

As NO is freely diffusible, in some instances there may be no need todeliver NO directly to targets, as some actions of NO are prevented byhemoglobin, which can sequester freely diffusible NO(21). We examinedthe effects of hemoglobin on S-nitrosylation of COX2 under varyingconditions. In HEK293T cells transfected with COX2, hemoglobin preventsthe S-nitrosylation elicited by GSNO (FIG. 9A) whereas it fails to alterS-nitrosylation of COX2 in RAW264.7 cells activated by LPS/IFNγ (FIG.9B). Thus in the more physiologic macrophage cell line, theS-nitrosylation of COX2 generated by an inflammatory stimulus does notappear to be elicited by freely diffusible NO.

EXAMPLE 5 S-Nitrosylation of COX2 Activates Enzyme Activity

To determine whether S-nitrosylation of COX2 alters enzyme activity, weexamined COX2 enzyme activity in HEK293T cells transfected withCOX2-myc. The NO donor SNP, added to cell lysates, elicits a substantialaugmentation of COX2 activity, which reflects S-nitrosylation, asascorbic acid, which reverses S-nitrosylation(20, 22), prevents theincrease (FIG. 2C and D).

The reversal by ascorbate of COX2 activation by NO donors is not merelya reflection of ascorbate influences on enzymes substrates orintermediate products, as ascorbate fails to affect COX2 activity inpreparations not treated by SNP. A relationship of S-nitrosylation andCOX2 activation is further supported by the closely similarconcentration-response relationship between the effects of the NO donorGSNO on S-nitrosylation and on COX2 activity (FIG. 2E).

NO activates COX2 by increasing its apparent Vmax without changing itsKm (FIG. 2E). The higher concentration of SNP required to activate COX2in vitro compared to intact cells accords with earlier studies showinggreater potency of NO donors in intact cells(23, 24). To ascertain thekinetic basis for NO activation of COX2, we conducted enzyme assays withincreasing concentrations of sucrose to augment viscosity and slow downenzyme kinetics (FIG. 2G). As expected, with increasing viscosity, theratio of control enzyme activity to the activity in more viscoussolutions increases. This increase is diminished in SNP samplesconsistent with SNP accelerating the release of product from the enzyme.

EXAMPLE 6 Identification of Cysteine Residue(s) Nitrosylated

We wondered which of the 13 cysteines of COX2 are critical for theaugmentation of COX2 activity elicited by S-nitrosylation. In RAW 264.7cells transfected with the N-terminal 483 amino acids or the C-terminal120 amino acids of COX2, LPS/IFNγ treatment leads to S-nitrosylation ofthe C-terminal fragment (which contains 3 cysteines) but not theN-terminal fragment (FIG. 10A). To ascertain which of these 3 cysteinesis responsible for augmented COX2 activity we mutated each of them toserine. The C526S mutation prevents activation of COX2 activity by theNO donor SNP, while the C561S mutation does not (FIG. 10B). The C555Smutation abolishes enzyme activity so the effects of NO stimulationcannot be assessed. Individual mutation of the 13 cysteines in COX2 doesnot detectably diminish total S-nitrosylation of the enzyme (data notshown), which suggests that multiple cysteines can be S-nitrosylated butonly C526 is responsible for enzyme activation by NO.

EXAMPLE 7 The Influence of NO on PGE₂ Formation

To clarify the influence of NO on PGE₂ formation in a more physiologicpreparation, we employed RAW264.7 cells. The formation of PGE₂ inresponse to LPS/IFNγ is inhibited by the iNOS inhibitor 1400W with 50%reduction of PGE₂ formation at drug concentrations which provide 50%inhibition of iNOS activity (FIG. 3A). Specificity of the NO associationis evident by inhibition of PGE₂ formation with the active L-isomer ofthe NOS inhibitor nitro-argininemethylester (L-NAME) but not by D-NAME;the effects of L-NAME are reversed by added L-arginine (FIG. 3B). Thus,about 50% of induced COX2 activity is determined by S-nitrosylation.

As RAW264.7 cells are a continuous macrophage cell line which may notbehave the same as macrophages in intact organisms, we also testedperitoneal macrophages obtained from iNOS knockout mice. PGE₂ formationfrom macrophages of LPS/IFNγ-treated mice is profoundly reduced in theiNOS knockout mice in parallel with a similar reduction in nitriteformation by the macrophages (FIG. 3C) and a decrease in S-nitrosylatedCOX2 (FIG. 3D). These observations concur with findings of decreasedurinary PGE₂ in iNOS knockout mice(25).

EXAMPLE 8 Fragment of COX2 (Amino Acids 484-604) Abolishes theCo-Precipitation of iNOS and COX2

We hypothesized that the augmentation of PGE₂ formation by iNOSactivation reflects binding of iNOS to COX2 to deliver NO in appropriateproximity for S-nitrosylation. To explore this possibility we utilizeddominant-negative constructs to block iNOS-COX2 binding using thefragment of COX2, amino acids 484-604, which binds iNOS (FIG. 4A).Transfection of 484-604 into RAW264.7 cells abolishes theco-precipitation of iNOS and COX2 and is associated with precipitationof 484-604 together with iNOS (FIG. 4A). Moreover, this interference ofbinding between COX2 and iNOS by 484-604 decreases S-nitrosylation ofCOX2 in RAW264.7 cells (FIG. 4B). The dominant-negative transfectionalso reduces PGE₂ formation by more than 50%, whereas transfection of afragment comprising amino acids 1-483, which does not bind iNOS, failsto influence PGE2 formation (FIGS. 4C and D).

REFERENCES

The disclosure of each reference cited is expressly incorporated herein.

-   -   1. M. E. Turini, R. N. DuBois, Annu. Rev. Med. 53, 35 (2002).    -   2. S. Moncada, J. R. Soc. Med. 92, 164 (1999).    -   3. R. J. Flower, Nat. Rev. Drug Discov. 2, 179 (2003).    -   4. D. Mukherjee, S. E. Nissen, E. J. Topol, JAMA 286, 954        (2001).    -   5. E. J. Topol, JAMA 293, 366 (2005).    -   6. T. P. Misko, J. L. Trotter, A. H. Cross, J. Neuroimmunol. 61,        195 (1995).    -   7. I. Appleton, A. Tomlinson, D. A. Willoughby, Adv. Pharmacol.        35, 27 (1996).    -   8. D. Salvemini et al., J. Clin. Invest 93, 1940 (1994).    -   9. J. R. Vane, Y. S. Bakhle, R. M. Botting, Annu. Rev.        Pharmacol. Toxicol. 38, 97 (1998).    -   10. D. Salvemini et al., Proc. Natl. Acad. Sci. U.S.A 90, 7240        (1993).    -   11. J. M. Braughler, C. K. Mittal, F. Murad, J. Biol. Chem. 254,        12450 (1979).    -   12. J. C. Edwards et al., Biochem. Pharmacol. 30, 2531 (1981).    -   13. J. S. Stamler, D. J. Singel, J. Loscalzo, Science 258, 1898        (1992).    -   14. S. R. Jaffrey, H. Erdjument-Bromage, C. D. Ferris, P.        Tempst, S. H. Snyder, Nat. Cell Biol. 3, 193 (2001).    -   15. D. T. Hess, A. Matsumoto, S. O. Kim, H. E. Marshall, J. S.        Stamler, Nat. Rev. Mol. Cell Biol. 6, 150 (2005).    -   16. R. M. Garavito, A. M. Mulichak, Annu. Rev. Biophys. Biomol.        Struct. 32, 183 (2003).    -   17. A. L. Tsai, C. Wei, R. J. Kulmacz, Arch. Biochem. Biophys.        313, 367 (1994).    -   18. T. A. Kennedy, C. J. Smith, L. J. Marnett, J. Biol. Chem.        269, 27357 (1994).    -   19. C. J. Smith, L. J. Mamett, Arch. Biochem. Biophys. 335, 342        (1996).    -   20. S. R. Jaffrey, S. H. Snyder, Sci. STKE. 2001, L1 (2001).    -   21. F. Murad, C. K. Mittal, W. P. Arnold, S. Katsuki, H. Kimura,        Adv. Cyclic. Nucleotide. Res. 9, 145 (1978).    -   22. Y. Yang, J. Loscalzo, Proc. Natl. Acad. Sci. U.S.A 102, 117        (2005).    -   23. J. Heo, S. L. Campbell, Biochemistry 43, 2314 (2004).    -   24. H. M. Lander, J. S. Ogiste, S. F. Pearce, R. Levi, A.        Novogrodsky, J. Biol. Chem. 270, 7017 (1995).    -   25. L. J. Marnett, T. L. Wright, B. C. Crews, S. R.        Tannenbaum, J. D. Morrow, J. Biol. Chem. 275, 13427 (2000).    -   26. D. Salvemini et al., J. Clin. Invest 96, 301 (1995).    -   27. M. Fang et al., Neuron 28, 183 (2000).    -   28. H. C. Kornau, L. T. Schenker, M. B. Kennedy, P. H. Seeburg,        Science 269, 1737 (1995).    -   29. R. L. Patterson, D. B. van Rossum, A. I. Kaplin, R. K.        Barrow, S. H. Snyder, Proc. Natl. Acad. Sci. U.S.A 102, 1357        (2005).    -   30. J. Roth, B. Storr, J. Goldbach, K. Voigt, E. Zeisberger,        Eur. J. Pharmacol. 383, 177 (1999).    -   31. H. Guhring et al., J. Neurosci. 20, 6714 (2000).    -   32. H. Krum, D. Liew, J. Aw, S. Haas, Expert. Rev. Cardiovasc.        Ther. 2, 265 (2004).

1. A method of screening for substances useful for relievinginflammation, comprising: contacting a test substance with a firstprotein and a second protein under conditions in which the first proteinand the second protein bind to each other, wherein the first protein isselected from the group consisting of mammalian iNOS, a fragment ofmammalian iNOS from the N-terminal 10% of iNOS sufficient to bind COX2,and a fusion protein comprising said fragment of mammalian iNOS, whereinthe second protein is selected from the group consisting of mammalianCOX2, a fragment of mammalian COX2 from the C-terminal 20% of COX2sufficient to bind iNOS, and a fusion protein comprising said fragmentof COX2; determining amount of free or bound of said first or saidsecond protein; identifying a test substance which increases the amountof free first or second protein or decreases the amount of bound firstor second protein as a candidate drug for relieving inflammation.
 2. Themethod of claim 1 wherein the step of contacting is done in vitro. 3.The method of claim 1 wherein the step of contacting is done in yeastcells containing recombinant forms of the first and second proteins. 4.The method of claim 3 wherein the first and second recombinant proteinsare each fused to a first and second yeast protein, wherein the firstand second yeast proteins reconstitute a functional transcriptionalactivator when brought into physical proximity by binding of the firstrecombinant protein to the second recombinant protein.
 5. The method ofclaim 1 further comprising the step of: testing an identified candidatedrug in an animal to determine if the candidate drug relievesinflammation in the animal.
 6. The method of claim 1 wherein the testsubstance is contacted with iNOS and COX2.
 7. The method of claim 1wherein the test substance is contacted with said fragment of mammalianiNOS and said fragment of mammalian COX2.
 8. The method of claim 1wherein the test substance is contacted with said fusion proteincomprising mammalian iNOS and said fusion protein comprising mammalianCOX2.
 9. The method of claim 1 wherein the test substance is contactedwith one of said mammalian proteins and one of said fragments.
 10. Themethod of claim 1 wherein the test substance is contacted with one ofsaid fusion proteins and one of said fragments.
 11. The method of claim1 wherein the test substance is contacted with one of said mammalianproteins and one of said fusion proteins.
 12. The method of claim 1wherein antibodies are used to determine said amount.
 13. The method ofclaim 1 wherein bound protein is determined by co-immunoprecipitation.14. The method of claim 1 wherein said step of contacting a testsubstance with a first protein and a second protein is accomplished bycontacting the test substance with a cell which comprises the firstprotein and the second protein.
 15. The method of claim 1 wherein themammalian iNOS fragment comprises amino acids 1-114 of human iNOS. 16.The method of claim 1 wherein the mammalian COX2 fragment comprisesamino acids 488-604 of human COX2.
 17. The method of claim 1 wherein themammalian COX2 fragment comprises amino acids 484-604 of murine COX2.